We propose to address the serious problem of non-adherence to pre-exposure HIV prophylaxis (PrEP), antiretroviral therapy (ART), and all medications including placebo in trials, by developing quantitative assays to assess adherence objectively using long half-life drug and taggant moieties. We propose two aims. First, we will take a well-established assay for assessing cumulative adherence to tenofovir (TFV)-based regimens and will bring it to the point of care. The assay is tenofovir-diphosphate (TFV-DP) in red blood cells (RBC), which we have shown to be a powerful predictor of adherence and therapeutic outcomes to TFV-based PrEP, because of its 17 day half-life. We introduce a novel point of care platform, the Mini 12 miniature mass spectrometer, developed by co-investigator Dr. Ouyang. The Mini 12 is the size of a shoebox and is designed for drug quantitation at the point of care through rapid analysis (minutes) of small blood volumes with simple steps and user-friendly interface so that unspecialized personnel can perform the assay. The estimated cost of the Mini 12 unit is $25K and the per-sample cost is $10 to $20, far less-expensive compared with lab-based assays. In developing the methodology, we will leverage existing whole blood samples from an ongoing directly observed dosing study to guide the transfer of the assay to the Mini 12. The leveraged samples arise from a study that is assessing different adherence patterns, enabling us to correlate TFV-DP measurements using the Mini 12 with adherence to TFV-based therapy. This aim will culminate in beta-testing the Mini 12 in the clinic. Secondly, to address non-TFV-based regimens, we present a new idea to use trace doses of inert stable labeled endogenous compounds for taggants and we provide rationale and preliminary data in support of the novel features of the idea, including the unique long-half kinetics based on turn-over rates and pool sizes, proven safety in all ages of humans, and promise for trace doses using +3 or higher stable isotopes (rather than +1 or +2) which prevents background interference from natural isotopes. The trace doses mean easier co- formulation with any drug or placebo and low likelihood of perturbing the steady-state system. Four taggants were identified for initial study. We will use the rat model to define taggants with suitable half-lives; 3 to 10 days in rats, scaling to 10 to 30 days in humans, similar in length to TFV-DP in DBS. Once the taggants are identified and defined in the rat model, the two best taggants will be pilot tested in humans to evaluate feasibility and scalability from the rat model to humans. This application will lay the groundwork for future studies to use the Mini 12 platform for taggants to ultimately extend this idea to the point of care. Taken together, these aims address the key objectives of this RFA-AI-14-071, to develop assays that verify drug ingestion, quantify adherence, and bring these tools to the bedside to enable real-time patient feedback.